Nonacetylated α-tubulin was obtained from HeLa S3 cells (American Type Culture Collection, CCL-2.2TM) for in vitro acetylation assays or from WT and Atat1 KO mouse brains for in vitro transport assay according to the protocol adapted from (45). Cells were lysed in BRB80 [80 mM K Pipes (pH 6.8), 1 mM MgCl2, and 1 mM EGTA] supplemented with 1 mM β-mercaptoethanol, 1 mM PMSF, and protease inhibitors at 4°C. The soluble fraction was obtained by ultracentrifugation, followed by tubulin polymerization at 30°C min by adding 1 mM guanosine triphosphate (GTP) and 30% glycerol. MTs were pelleted by ultracentrifugation at 30°C for 30 min and depolymerized in BRB80 at 4°C; soluble tubulin was clarified by ultracentrifugation at 4°C. The second polymerization round was performed for 30 min at 30°C in the presence of high-molarity Pipes to remove mitogen-activated proteins; the MT pellet was sedimented by ultracentrifugation. MTs were then depolymerized in BRB80 at 4°C, and soluble tubulin was clarified and snap-frozen.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.